Description
Malate Dehydrogenase Assay (Colorimetric) (BA0131) (BA0131)
The Malate Dehydrogenase Assay (Colorimetric) (SKU: BA0131) provides a rapid, non-radioactive method for quantifying malate dehydrogenase (MDH; EC 1.1.1.37) activity in biological samples. MDH reversibly catalyses the oxidation of L-malate to oxaloacetate in the presence of NAD, and exists as two isoforms, the cytoplasmic MDH1 and the mitochondrial MDH2, the latter participating in the TCA cycle. Elevated MDH activity has been associated with certain neurodegenerative conditions such as Alzheimer's disease. In this assay, the reduction of the tetrazolium salt MTT in an NADH-coupled enzymatic reaction produces a reduced form of MTT with an absorption maximum at 565 nm, and the increase in absorbance is proportional to the enzyme activity. The homogeneous mix-incubate-measure format is convenient and can be readily automated for high-throughput screening. The linear detection range is 0.5 to 65 U/L for a 20 minute reaction at 37 degrees C.
| Product Name: | Malate Dehydrogenase Assay (Colorimetric) (BA0131) |
| SKU: | BA0131 |
| Detection Method: | Colorimetric |
| Detection Range: | 0.5 to 65 U/L (20 uL sample, 20 min reaction at 37 degrees C) |
| Sample Type: | Plasma, serum, erythrocytes, tissue, cell lysate and culture media |
| Species Reactivity: | All |
| Assay Time: | 30 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20 degrees C |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
This kit measures malate dehydrogenase activity based on the reduction of the tetrazolium salt MTT in an NADH-coupled enzymatic reaction to a reduced form of MTT, which exhibits an absorption maximum at 565 nm. The increase in absorbance at 565 nm is proportional to the enzyme activity, allowing straightforward kinetic quantitation of MDH.
- Fast and sensitive with a linear detection range of 0.5 to 65 U/L (20 uL sample) for a 20 minute reaction at 37 degrees C
- Convenient and high-throughput homogeneous mix-incubate-measure type assay
- Can be readily automated on HTS liquid handling systems for processing thousands of samples per day
- MDH activity determination in biological samples such as plasma, serum, erythrocytes, tissue and culture media
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Equilibrate reagents to the desired reaction temperature (37 degrees C is recommended) and briefly centrifuge tubes before use. |
| 2 | Transfer 100 uL H2O and 100 uL Calibrator solution into wells of a clear flat-bottom 96-well plate. |
| 3 | Transfer 20 uL H2O into one well to serve as the blank, and transfer 20 uL of each sample into separate wells. |
| 4 | Prepare sufficient Working Reagent for all reaction wells by mixing, for each 96-well assay, 74 uL Assay Buffer, 8 uL NAD/MTT, 5 uL Substrate, 1 uL Enzyme A and 1 uL Enzyme B. |
| 5 | Add 80 uL Working Reagent to all sample and blank wells and tap the plate briefly to mix. |
| 6 | Read OD565nm at time 10 minutes and time 30 minutes on a plate reader. |
Subtract the OD10 from OD30 for each sample to compute the delta ODS values, and do the same for the blank to compute delta ODB. MDH Activity = [(delta ODS - delta ODB) / (ODCAL - ODH2O)] x (273 / t) x n (U/L), where t is the difference in time between readings (20 minutes is the recommended time at 37 degrees C), ODCAL and ODH2O are OD565nm (OD10) values of the Calibrator and water, and n is the dilution factor. One Unit (U) of MDH will catalyse the conversion of 1 umole of oxaloacetate and NADH per minute at pH 7.5.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20 degrees C |
| NAD/MTT | 1 mL | -20 degrees C |
| Substrate | 600 uL | -20 degrees C |
| Enzyme A | 120 uL | -20 degrees C |
| Enzyme B | 120 uL | -20 degrees C |
| Calibrator | 1.5 mL | -20 degrees C |
